Summary of Work: Defects in the two major pathways of double-strand break (DSB) repair, homologous recombination and nonhomologous end- joining (NHEJ), are implicated in several human cancers and aging. We have focused on DSB repair in yeast using in vivo systems to generate defined DSBs. The role of genes involved in recombination, NHEJ, nucleotide excision repair and cell cycle checkpoint responses were investigated. Accurate NHEJ repair is efficient, particularly for DSBs related to those found during cell development. Ionizing radiation, methyl methanesulfonate (MMS), and site-specific endonucleases revealed differences between genes needed for DSB repair. For example, cells lacking the Rad50/Mre11/ Xrs2 deoxyribonuclease complex are more sensitive to endonuclease- and MMS-induced cell killing than cells deficient in other repair pathways. rad50, mre11 and xrs2 mutants were no more gamma radiation sensitive than recombination- deficient cells, suggesting that DSB end structure may determine repair mechanisms. A search for high-copy suppressors of rad50, mre11 and xrs2 mutant MMS- sensitivity identified EXO1, a 5?>3? exonuclease associated with recombination and mismatch repair, and TLC1, the RNA template component of the yeast telomerase complex. We initiated a study with Kerry Bloom at UNC (also DOE supported) to combine genetic and cytological approaches to characterize DSB induced chromosomal changes and repair in real-time. Recently developed techniques are being used to monitor movements of yeast chromosomes and associated structures in living cells. Digital- and video-enhanced (DE and VE) differential interference contrast (DIC) microscopic imaging techniques, are use to monitor the behavior of green fluorescent protein (GFP)-tagged broken DNA ends after specific DNA cleavage. Interactions of broken DNA molecules with each other and with the mitotic spindle can be followed in normal and repair defective cells. This will provide the first opportunity to assess the relationship between molecularly and cytologically detected breaks and the associated genetic factors. - Molecular, DNA Damage, DNA Repair, Genetic Vectors, Ionizing, Meiosis, Methyl Methane Sulfonate, Radiation, Ultraviolet light